The corneal stroma contains keratocytes imbedded in an extracellular matrix consisting primarily of collagen types I and V and of proteoglycans that contain either chondroitin sulfate (CS) or keratan sulfate (KS) chains. Electron microscopic studies show the corneal stroma contains collagen fibrils of small, uniform diameter that are separated by small, uniformly sized spaces. The collagen fibrils in the corneal stroma are heterofibrils of collagen types I and V. Collagen type V is essential for the initiation of fibril formation and the presence of collagen V in the heterofibril has been shown to limit the fibril diameter growth. The proteoglycans are in the spaces between the fibrils in vivo (see Muller L J, Pels E, Schurmans L R, and Vrensen G F. A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma Exp Eye Res. 2004; 78:493-501; which is incorporated herein by reference) and in vitro assays that measure collagen fibril assembly have shown that these proteoglycans modulate collagen fibril formation. Collagen fibril formation in the presence of CS and KS proteoglycans purified from the cornea delayed fibril formation, decreased the rate of fibril growth and resulted in smaller collagen fibrils. The removal of the GAG side chains did not affect the activity of the proteoglycans, but reduction and alkylation abolished the activity. This indicates that the core protein of these proteoglycans modulates collagen assembly into fibrils. The major proteoglycans of the corneal stroma are decorin, lumican and keratocan. Decorin is a CS proteoglycan, whereas keratocan and lumican are KS proteoglycans. Keratocan (see Iu C Y, Birk D E, Hassell J R, Kane B, and Kao W W. Keratocan-deficient mice display alterations in corneal structure J Biol Chem. 2003; 278:21672-21677; Meek K M, Quantock A J, Boote C, Liu C Y, and Kao W W. An X-ray scattering investigation of corneal structure in keratocan-deficient mice Matrix Biol. 2003; 22:467-475; which are incorporated herein by reference) and lumican (see Chakravarti S, Petroll W M, Hassell J R, et al. Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma Invest Ophthalmol Vis Sci. 2000; 41:3365-3373; Chakravarti S, Magnuson T, Lass J H, et al. Lumican regulates collagen fibril assembly: skin fragility and corneal opacity in the absence of lumican J. Cell Biol. 1998; 141:1277-1286; Chakravarti S. Functions of lumican and fibromodulin: lessons from knockout mice Glycoconj J. 2002; 19:287-293 which are incorporated herein by reference) null mice have thinner corneas, and the collagen fibrils in the stromas are larger and less organized than in the stromas of normal mice, confirming the in vitro turbidimetry analysis of collagen fibril assembly.
Three procollagen polypeptides come together to form a left-handed triple helix immediately after synthesis. Stable triple helix formation, however, can only occur if certain lysine and proline residues in the collagen molecule are hydroxylated. This post-translational hydroxylation is performed by either lysyl or prolyl hydroxylases. These enzymes are found in the lumen of the endoplasmic reticulum and require ascorbic acid as a cofactor. Ascorbate deficiency in cell culture does not affect collagen synthesis but affects fibril formation and the rate of collagen secretion. The unhydroxylated collagen molecules denature at a lower temperature and in less stringent environments than properly hydroxylated collagen fibrils. Systemic ascorbate deficiency leads to scurvy, and this deficiency affects wound healing. Scorbutic wounds are weaker than non-scorbutic wounds, prone to reopen and contain a mass of irregular, unorganized collagen fibrils.
The cornea and anterior segment of the eye contain high levels of ascorbate (see Ringvold A, Anderssen E, and Kjonniksen I. Distribution of ascorbate in the anterior bovine eye Invest Ophthalmol Vis Sci. 2000; 41:20-23; which is incorporated herein by reference), where it is thought to function as an antioxidant and protect the cornea from reactive oxygen species that result from UV irradiation (see Brubaker R F, Bourne W M, Bachman L A, and McLaren J W. Ascorbic acid content of human corneal epithelium Invest Ophthalmol Vis Sci. 2000; 41:1681-1683; which is incorporated herein by reference). The levels of ascorbate in the aqueous humor and the cornea drop following an alkali burn, resulting in the cornea becoming scorbutic. The cells in the stroma show characteristics of scorbutic tissue such as a sparse ER, suggesting that they are not very metabolically active (Pfister R R, and Paterson C A. Ascorbic acid in the treatment of alkali burns of the eye Ophthalmology. 1980; 87:1050-1057; which is incorporated herein by reference). Topical ascorbate has been used successfully to treat corneal alkali burns and has been proposed to do so by increasing the synthesis and secretion of properly folded collagen to replace the collagen denatured by the burn (see Pfister R R, and Paterson C A. Additional clinical and morphological observations on the favorable effect of ascorbate in experimental ocular alkali burns Invest Ophthalmol Vis Sci. 1977; 16:478-487; which is incorporated herein by reference). In addition, topical ascorbate has also been used post-photorefractive keratectomy (PRK) where its use decreased the late onset corneal haze (see Stojanovic A, Ringvold A, and Nitter T. Ascorbate prophylaxis for corneal haze after photorefractive keratectomy J Refract Surg. 2003; 19:338-343; which is incorporated herein by reference). Ascorbate concentration is highest in the epithelium, the layer that is removed prior to PRK. While the exact reason for the late onset corneal haze is not known, it is possible that removal of part of the epithelium prior to PRK results in a drop in ascorbate levels in the anterior corneal stroma and we would further speculate that this drop may reduce the secretion of properly folded collagen which could result in the haze.
Ascorbic acid has been used to study collagen synthesis in culture, but it is easily oxidized in solution and its metabolic by-products are cytotoxic in extended cell culture. A stable, non-toxic phosphate derivative of ascorbic acid (2-phospho-L-ascorbic acid) was developed and has been shown to stimulate collagen accumulation by skin fibroblasts in culture and also to enhance the secretion of type I and type III collagen peptides by rabbit keratocytes cultured in medium containing fetal bovine serum. Serum, however, contains mitogens and morphogens that cause keratocytes in culture to proliferate, acquire a fibroblastic morphology and cease keratocan expression.